Nucleic acid detection with CRISPR-Cas13a/C2c2

Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Lee, Jeong Wook; Essletzbichler, Patrick; Dy, Aaron J.; Joung, Julia; Verdine, Vanessa K.; Donghia, Nina M.; Daringer, Nichole M.; Freije, Catherine A.; Myhrvold, Cameron; Bhattacharyya, Roby P.; Livny, Jonathan; Regev, Aviv; Koonin, Eugene V.; Hung, Deborah T.; Sabeti, Pardis C.; Collins, James J.; Zhang, Feng

doi:10.1126/science.aam9321

Cited by 2,713 publications

(3,042 citation statements)

References 33 publications

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“…This HEPN-activated conformation of Cas13 is a general nuclease, which is capable of cleaving either the RNA molecule that it was activated by ( cis cleavage), or any other RNA molecule that it happens to encounter ( trans / collateral cleavage). All Cas13 proteins characterized to date exhibit these behaviors (Abudayyeh et al, 2016; East-Seletsky et al, 2016; East-Seletsky et al, 2017; Gootenberg et al, 2018; Gootenberg et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). …”

Section: Introductionmentioning

confidence: 99%

“…The RNA-activated RNA cleavage behavior of Cas13 provides a mechanism for RNA detection and diagnostic applications (East-Seletsky et al, 2016; Gootenberg et al, 2018; Gootenberg et al, 2017), as well as RNA-targeting in heterologous systems (Abudayyeh et al, 2017; Aman et al, 2018; Cox et al, 2017; Konermann et al, 2018). Versions of Cas13 where the HEPN-domains have been catalytically inactivated (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018) (deactivated Cas13; dCas13) have shown to be useful as programmable RNA binding proteins for RNA imaging (Abudayyeh et al, 2017), RNA editing (Cox et al, 2017) and regulating specific transcripts RNA in other ways (e.g.…”

Section: Introductionmentioning

confidence: 99%

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RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

et al. 2018

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SUMMARY CRISPR-Cas13a enzymes are RNA-guided, RNA-activated ribonucleases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging and RNA regulation. However, the relationship between target RNA binding and HEPN (higher-eukaryotes-and-prokaryotes nucleotide-binding)- domain nuclease activation is not well understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a’s target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number of and position of mismatches between the guide and target. We identify a central ‘binding seed’ where perfect base pairing is absolutely required for target binding, and a separate ‘nuclease switch’ where imperfect base-pairing results in tight binding but no HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a’s RNA binding and cleavage behavior for RNA-targeting tool development.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

et al. 2018

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“…The RNA-targeting CRISPR-associated enzyme Cas13(1, 2) has recently been adapted for such purpose. This detection platform, termed SHERLOCK ( S pecific H igh Sensitivity E nzymatic R eporter Un LOCK ing) (3), can discriminate between inputs that differ by a single nucleotide at very low concentrations and can be lyophilized for portable deployment. However, this technology has several limitations, including the lack of quantitation and reliance on fluorescent detection equipment for readout.…”

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confidence: 99%

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

Gootenberg

Abudayyeh

Kellner

et al. 2018

Science

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Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.

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“…8 Because it is desirable to optimize detection speed, sensitivity, selectivity, and cost for bacterial diagnostics, research into bacterial detection continues to be of substantial interest. 5,9–11 …”

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confidence: 99%

Whole-Cell Pseudomonas aeruginosa Localized Surface Plasmon Resonance Aptasensor

Bohn

2018

Anal. Chem.

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The detection of whole-cell Pseudomonas aeruginosa presents an intriguing challenge with direct applications in health care and the prevention of nosocomial infection. To address this problem, a localized surface plasmon resonance (LSPR) based sensing platform was developed to detect whole-cell Pseudomonas aeruginosa strain PAO1 using a surface-confined aptamer as an affinity reagent. Nanosphere lithography (NSL) was used to fabricate a sensor surface containing a hexagonal array of Au nanotriangles. The sensor surface was subsequently modified with biotinylated polyethylene glycol (Bt-PEG) thiol/PEG thiol (1:3), neutravidin, and biotinylated aptamer in a sandwich format. The 1:3 (v/v) ratio of Bt-PEG thiol/PEG thiol was specifically chosen to maximize PAO1 binding while minimizing nonspecific adsorption and steric hindrance. In contrast to prior whole-cell LSPR work, the LSPR wavelength shift was shown to be linearly related to bacterial concentration over the range of 10–103 cfu mL−1. This LSPR sensing platform is rapid (~3 h for detection), sensitive (down to the level of a single bacterium), selective for detection of Pseudomonas strain PAO1 over other strains, and exhibits a clinically relevant dynamic range and excellent shelf life (≥2 months) when stored at ambient conditions. This versatile LSPR sensing platform should be extendable to a wide range of supermolecular analytes, including both bacteria and viruses, by switching affinity reagents, and it has potential to be used in point-of-care and field-based applications.

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Nucleic acid detection with CRISPR-Cas13a/C2c2

Cited by 2,713 publications

References 33 publications

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

Whole-Cell Pseudomonas aeruginosa Localized Surface Plasmon Resonance Aptasensor

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